The present invention relates to adenoviral vectors which contain a cassette for expressing a gene of interest which is placed under the control of the elements which are required for expressing it, and which comprise splicing sequences. The presence of these sequences markedly increases the expression of the therapeutic gene in a host cell or organism. The invention also relates to the cells and the infectious viral particles which contain these novel vectors and to a method for preparing them. The invention is of especial interest from the point of view of gene therapy, in particular gene therapy in man.
Gene therapy is defined as being the transfer of genetic information into a host cell or organism. The first protocol applied to man was initiated, in the United States in September 1990, on a patient who was genetically immunodeficient due to a mutation which affected the gene encoding Adenine Deaminase (ADA). The relative success of this first experiment encouraged the development of this technology for a variety of diseases including both genetic (with the aim of correcting the malfunction of a defective gene) and acquired (infectious diseases, cancers, etc.) diseases. At the present time, the majority of protocols use retroviral vectors for transferring the therapeutic gene into the cells to be treated and expressing it in the cells. However, in addition to their restricted cloning capacity, retroviral vectors suffer from two major drawbacks which limit their systematic use: on the one hand, they mainly infect dividing cells and on the other hand, because they integrate randomly into the genome of the host cell, the risk of insertional mutagenesis is not insignificant. For this reason, a large number of scientific groups has been endeavoring to develop other types of vector, including adenoviruses.
Adenoviruses have been demonstrated to be present in a large number of animal species; they are not very pathogenic, they do not integrate and they replicate equally well in dividing and quiescent cells. Furthermore, they have a wide host spectrum and are able to infect a large number of cell types, in particular epithelial and endothelial cells, myocytes, hepatocytes, nerve cells and synoviocytes. In addition, they possess a natural tropism for the airways. These specific properties make adenoviruses vectors of choice for a large number of therapeutic and even vaccinal applications.
As a general rule, the adenoviral genome consists of a linear, double-stranded DNA molecule of approximately 36 kb in size which carries more than 30 genes encoding the viral proteins and, at its ends, two inverted repeats (designated ITR, standing for Inverted Terminal Repeat) and the encapsidation region. The early genes, which are required for replicating the virus, are divided up into 4 regions (E1 to E4, E for early) which are dispersed within the genome and which contain 6 transcriptional units provided with their own promoter. The late genes (L1 to L5, L for late), which encode the structural proteins, partially cover the early transcription units and are for the most part transcribed from the major late promoter MLP (see FIG. 1).
The adenoviral vectors which are currently employed in gene therapy protocols are so-called first-generation vectors, which vectors lack the major part of the E1 region, which is essential for replication, in order to avoid the vectors being disseminated in the environment and in the host organism. Deletion of the non-essential E3 region increases the cloning capacity of the vectors. The genes of interest are introduced into the viral DNA in place of one or other of the deleted regions. These replication-defective viruses can be propagated in a cell line which complements the E1 function. Use is commonly made of the 293 cell line, which was developed from human embryonic kidney cells (Graham et al., 1977, J. Gen. Virol. 36, 59-72). The deletion of the non-essential E3 region does not require any specific complementation. Even if the feasibility of transferring genes using these first-generation vectors is now well established, there is still the question of whether or not they are harmless. As well as the safety aspects (risk of generating replication-competent particles), the problem of their toxicity also arises. Thus, the first clinical trials provided evidence of the induction of inflammatory responses which were due to the expression of the viral genes in the host and which opposed the persistence of the transduced cells and expression of the transgene. These drawbacks linked to stimulation of the host immune system by the adenoviral epitopes have justified constructing new-generation viruses.
The design of an adenoviral vector is on the one hand based on the viral skeleton and on the other hand based on the cassette for expressing the therapeutic gene, with this gene being combined with regulatory elements which enable it to be expressed optimally in the host cell. With regard to the first point, the second-generation adenoviral vectors retain the in cis ITR regions and encapsidation sequences and contain substantial internal deletions which are aimed at suppressing most of the viral genes whose expression in vivo is undesirable (see international application WO94/28152). Their propagation is ensured by using a helper virus or cell lines which complement the defective functions. For example, a cell line which is derived from 293 and which expresses the adenoviral sequences encoding the essential E4 proteins will be used for complementing a second-generation vector whose genomic skeleton has been deleted for the E1, E3 and E4 regions.
As far as the expression cassette is concerned, this generally comprises a 5xe2x80x2 promoter region which directs transcription of the gene which follows it and possibly a 3xe2x80x2 polyadenylation (polyA) sequence which contributes, in particular, to stabilizing the transcribed messenger. Additional elements may improve expression under certain circumstances. The positive effect of intron sequences on gene expression has already been reported in vitro (Buchman and Berg, 1988, Mol. Cell. Biol. 8, 4395-4405; Huang and Gorman, 1990, Nucleic Acid Res. 18, 937-947), in vivo in transgenic animals (Brinster et al., 1988, Proc Natl. Acad. Sci. USA 85, 836-840) and, more recently, in the context of a first-generation adenoviral vector (Connelly et al., 1996, Human Gene Therapy 7, 183-195). This document shows that mice which have been treated with an adenovirus which has been deleted for the E1 and E3 regions and which is expressing human factor VIII produce levels of serum factor VIII which are 3 to 13 times higher when the complementary FVIII DNA contains splicing sequences.
The object of the present invention is to make adenoviral vectors available to the public which are more efficient from the point of view of expressing the therapeutic gene and which thereby make it possible to reduce the vector doses and to amplify the therapeutic effect. It has now been demonstrated that the presence of splicing sequences within the gene of interest is beneficial, if not essential, for obtaining expression of the gene. This observation is particularly true within the context of a second-generation adenoviral vector, where the levels at which the canine factor IX (FIX) and human interleukin 2 (IL-2) therapeutic genes are expressed are amplified by a factor of 20 to 150 when the expression cassette includes the said splicing sequences. The amplification factor remains significant (2 to 3) when a first-generation vector is used. This substantial improvement in gene expression is unexpected and could not be deduced from the state of the art.
For this reason, the present invention relates to an adenoviral vector which is derived from an adenovirus genome by deleting at least all or part of the E1 region, with the said adenoviral vector containing a cassette for expressing a gene of interest which is placed under the control of the elements which are required for expressing it in a host host cell or organism, with the said elements which are required for the expression comprising at least one splicing sequence, characterized in that the said splicing sequence is derived from a eukaryotic nuclear gene which is selected from the mammalian factor VIII, collagen, xcex1- or xcex2-globin and ovalbumin genes, or from a synthetic splicing sequence.